Globally, 350 million people are chronically infected with hepatitis B virus (HBV) - the world's leading cause of cirrhosis and hepatocellular carcinoma (HCC). Chronic HBV infection is characterized by the presence of hepatitis B surface antigen (HBsAg) in the serum. In contrast, occult HBV infection (O-HBV) is defined as low level HBV replication in the absence of detectable circulating HBsAg. The transmissibility of O-HBV and the subsequent establishment of chronic HBV infection are well documented in humans and in primates. Moreover, O-HBV is associated with advanced liver fibrosis, reduced response to interferon therapy, the development of HCC, and increased liver enzyme levels. In HIV-positive cohorts, the prevalence of O-HBV infection is significantly elevated. In South Africa, both HIV and HBV are endemic, and HIV co-infection is a major risk factor for O-HBV infection. Moreover, treatment resistance mutations in the HBV Polymerase (P) gene - which overlaps with the Surface (S) gene - are common in South Africa - even among treatment-naove individuals - and may also impact HBsAg expression. Only a small number of studies evaluated the potential mechanism(s) for this lack of HBsAg detection in vitro. Major limitations to characterizing O-HBV infection include 1) the lack of sensitive quantitative assays for HBV DNA, and 2) the limited ability to identify and characterize mutations that are associated with O-HBV in the context of full-length, replication-competent genomes. Fortunately, highly sensitive, cost effective real-time PCR assays for HBV DNA quantification - such as that developed within our lab - are now available. Moreover, a novel method for efficient amplification of whole HBV genomes that also permits rapid functional analysis has been developed. The aims of this application are to determine the effects of S and P gene mutations associated with O-HBV infection on HBsAg synthesis/retention/secretion and HBV replication in hepatocytes using full-length, replication-competent HBV expression vectors. The identification and characterization of O-HBV mutations in vivo that are not detected using current HBV screening assays and the development of in vitro assays to evaluate the mechanism(s) underlying the lack of HBsAg detection would improve future diagnostic sensitivity for O-HBV infection, limit secondary transmission of HBV, provide critical information on treatment options for O-HBV, and improve our understanding of how HBV replication contributes to the development of HCC.